The desirability of modifying biologically active and therapeutically useful polypeptides with a variety of compounds, such as the hydrophilic polymer polyethylene glycol (PEG), to enhance their pharmacokinetic properties has been noted. See, e.g., the discussion of the art in this area of polypeptide modification in published PCT patent application WO87/00056, in U.S. Pat. No. 4,179,337, which discloses conjugating water soluble polypeptides such as enzymes and insulin to PEG or PPG, and in U.S. Pat. No. 4,766,106, which discloses conjugating ordinarily water insoluble beta-interferon, interleukin-2, or immunotoxins to PEG homopolymers or polyoxyethylated glycerol. Such modification can reduce adverse immune response to the polypeptide, increase the solubility for use in pharmaceutical preparations and maintain a desirable circulatory level of such polypeptide for therapeutic efficacy.
One problem not addressed by the art in this area involves the extent to which a polypeptide can be modified by attachment of compounds having reactive groups that will covalently bond to certain amino acid residues of the polypeptide For example, modification of a polypeptide with PEG or similar polymers, can result in random attachment of the polymer at the amino terminus of the polypeptide and/or at one or more lysine residues in the amino acid sequence of the protein Because more than one PEG group can attach to the polypeptide, the resultant composition may contain a heterogeneous mixture of "PEGylated" polypeptide; some polypeptides having only one PEGylated site, others having more than one PEGylated site. Such heterogeneity in composition is undesirable for pharmaceutical use. Furthermore, the non-specificity with regard to the site(s) of attachment of compounds such as PEG to the polypeptide can result in loss of biological efficacy of the polypeptide stemming from undesirable attachment to a polypeptide site required for biological activity.
Co-pending, commonly owned U.S. patent application Ser. No. 137,043 now U.S. Pat. No. 4,904,584 entitled SITE-SPECIFIC HOMOGENEOUS MODIFICATION OF POLYPEPTIDES addresses the foregoing by providing materials and methods for site specific covalent modification of polypeptides by lysine insertion, removal, and/or replacement. However, we have determined that the use of lysine as the attachment site for modification, for example, by PEGylation, may be disadvantageous in the case of IL-3 because not all modifications may result in biologically active compounds and because steps must be taken to prevent PEGylation at N-termini in cases where N-terminal PEGylation is not desired.